G. L. Gianfranceschi

Evidence for the presence in calf thymus of a peptidic factor controlling DNA transcription in vitro

A thymic factor causes a strong inhibition of the DNA-directed RNA polymerase reaction in vitro. The active factor was isolated from aqueous ultrafiltered thymus extracts and purified by means of chromatography on DEAE-cellulose and then on Dowex 50 WX2. The purified thymic factor was characterized as a peptide of low molecular weight (less than 5000). The biological activity of the thymic factor cannot be attributed to the presence of a nuclease or of a histone fragment. The RNA synthesis is controlled by this factor by means of electrostatic interactions between the peptide compound and DNA. Inhibitory activity on RNA synthesis was absent from kidney extracts.

Small peptides controlling transcription in vitro are bound to chromatin DNA.

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 micrograms/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.

Low molecular weight peptides controlling transcription are present in the calf thymus chromatin structure

A calf thymus peptide fraction controlling DNA and chromatin template has been purified by DNA-cellulose and Dowex 50 WX2 chromatography and its amino acid composition determined. The active peptide fraction can be extracted in high pH buffer from calf thymus native chromatin previously deproteinized by chloroform-isoamylalcohol and phenol. These data demonstrate that the thymic peptide(s) is (are) a chromatin protein constituent strongly linked to DNA. The specificity in association of the peptide(s) to DNA has also been considered.

Stabilization of double-stranded DNA molecule by nonhistone peptidic effector from calf thymus

A peptidic effector from calf thymus causes a strong stabilization of DNA doublestranded molecule in vitro. The active factor was isolated from aqueous ultrafiltered thymus extracts and purified by means of chromatography on DEAE-cellulose and then on Dowex 50 WX2. The purified thymic factor was characterized as a peptide of low molecular weight (<5000). The biological activity of the thymic factor cannot be attributed to a histone fragment. Melting data of the control DNA and of the DNA-active factor complex in various conditions of ionic strength and dielectric constant of the solution medium are recorded.

Effects of a chromatin low molecular weight peptidic fraction on differentiation markers and virus production in Friend leukemia cells.

Low molecular weight chromatin peptides exert a dose-dependent inhibition of Dimethylsulfoxide (DMSO)-induced erythroid differentiation of murine Friend Leukemia Cells (FLC). This effect correlates with the degree of purification of the peptide fractions. Crot analysis of globin mRNA amounts in DMSO-treated FLC given the peptides showed a 4-5-fold decrease of messenger RNA in the cytoplasm with no nuclear storage of globin transcripts. Spectrin accumulation in “induced” FLC is inhibited as well. The effects of the peptides on erythroid markers are reversible upon removal of the compounds. They also appear to bespecific forinduced gene expression as (1) no effects are observed on cell growth and RNA synthesis in normalnondifferentiating cell lines; and (2) no changes have been detected with regard to the expression of integrated viral genes coding for continuous shedding of viral particles.

Control of RNA and protein synthesis in phytohemagglutinin-stimulated human lymphocytes by chromatin peptides from calf thymus.

Low molecular weight peptides from calf thymus chromatin inhibit RNA and protein synthesis in Phytohemagglutinin-stimulated human lymphocytes. These results may be correlated to the controlling transcription activity by chromatin peptides in cellfree systems.

Small acidic peptides are bound to E. coli DNA

Low molecular weight peptides have been isolated by alkali extraction from deproteinized DNA of E. coli cells grown in the presence of radioactive glutamic acid or orthophosphate. The labeled peptides, purified by gel filtration chromatography on Sephadex G25 and G10, contain prevailingly glutamic acid, aspartic acid, glycine, serine and alanine. Electrophoretic studies at different pH show that some peptide fractions contain a phosphoric residue. The N-terminus of the phosphorylated peptides is apparently blocked and they were able to bind to DNA in the presence of Mg2+ ions. Moreover the acidic peptides extracted from E. coli DNA show a sharp activity in the control of λ phage DNA transcription ‘in vitro’.

Specific thymic peptides-DNA interaction. Correlation with the possible stereochemical kinking scheme of DNA

Low molecular weight peptides from calf thymus cause a strong dose-dependent stabilization of the DNA. The strength of DNA-peptide interaction is pH-dependent and decreases rapidly above pH 6.5. Moreover the complete kinetics of DNA denaturation and renaturation demonstrates that the peptide fraction increases significantly the DNA renaturation mostly at low temperature, showing that the interaction DNA-thymic effector helps the recombination of complementary DNA segments. The DNA stabilization rate by the peptide fraction is comparable to that obtained by means of high concentration of histones or synthetic polycationic peptides. However, the lack of basic amino acids in the peptide structure is not in favor of strong electrostatic interactions and implies a specific binding of peptide to DNA. The possible correlation of the specific thymic peptides-DNA interaction with the stereochemical kinking scheme of DNA is discussed.

DNA-binding peptides from rat liver and Novikoff hepatoma cells: quantitative level and possible biochemical differences

DNA isolated from rat liver by intensive deproteinization with chloroform/isoamyl alcohol and phenol contains low molecular weight peptides in a quantity of about 20 μg/mg DNA.These peptides show high specific activity in inhibiting transcription in a reconstituted cell-free system with prokaryotic and eukaryotic RNA polymerase. Their level is markedly decreased in DNA prepared from Novikoff hepatoma cells. Moreover the amino acid analysis and the pattern of analytical separation by high performance liquid chromatography (HPLC) show some biochemical differences between DNA-binding peptides extracted from rat liver and Novikoff hepatoma cells. The possibility that carcinogenesis may involve mechanisms which lead to selective removal of some components of the DNA-binding peptides, is discussed.

Isolation and characterization of DNA-binding peptides from the serum: inhibition of transcription and comparison with the tissue peptides.

Small peptides highly active in inhibiting DNA transcription ‘in vitro’ by eukaryotic and prokaryotic RNA polymerases and in increasing the melting point of double stranded DNA can be isolated from the serum of various sources. Peptides with the same biological activity and with identical or very similar amino acid composition are linked to chromatin DNA of several tissues.